Hydrogels are continuously gaining increased attention as biomaterials for biomedical applications, such as tissue engineering and therapeutics delivery. Furthermore, in situ forming hydrogels or those exhibiting the specific ability of increasing their viscosity with temperature, also called thermosensitive, are preferred over preformed hydrogels, since cells and bioactive compounds, such as drugs, may be easily mixed with the precursor solutions prior to gelation to give homogeneously loaded gels. In addition, in situ gelation facilitates the application and allows for minimally invasive surgery and for adequately fill complex shaped lesion cavities.
Chitosan is an amino polysaccharide obtained by partial to substantial alkaline N-deacetylation of chitin also named poly(N-acetyl-D-glucosamine), which is a naturally occurring biopolymer found in exoskeleton of crustaceans, such as shrimp, crab and lobster shells. Chitosan contains free amine (—NH2) groups and may be characterized by the proportion of N-acetyl-D-glucosamine units and D-glucosamine units, which is expressed as the degree of deacetylation (DDA) of the fully acetylated polymer chitin. The properties of chitosan, such as the solubility and the viscosity, are influenced by the degree of deacetylation (DDA), which represents the percentage of deacetylated monomers, and the molecular weight (Mw).
Chitosan has been proposed in various formulations, alone and with other components, to stimulate repair of dermal, corneal and hard tissues in a number of reports (U.S. Pat. Nos. 4,572,906; 4,956,350; 5,894,070; 5,902,798; 6,124,273; and WO 98/22114). The properties of chitosan that are most commonly cited as beneficial for the wound repair process are its biodegradability, adhesiveness, prevention of dehydration and as a barrier to bacterial invasion. The interesting haemostatic potential of chitosan has also led to its direct application to reduce bleeding at grafts and wound sites (U.S. Pat. No. 4,532,134). Some studies claim that the haemostatic activity of chitosan derives solely from its ability to agglutinate red blood cells while others believe its polycationic amine character can activate platelets to release thrombin and initiate the classical coagulation cascade thus leading to its use as a haemostatic in combination with fibrinogen and purified autologous platelets (U.S. Pat. No. 5,773,033).
One technical difficulty that chitosan often presents is a low solubility at physiological pH and ionic strength, thereby limiting its use in a solution state. Thus typically, dissolution of chitosan is achieved via the protonation of amine groups in acidic aqueous solutions having a pH ranging from 3.0 to 5.6. Such chitosan solutions remain soluble up to a pH near 6.2 where neutralisation of the amine groups reduces interchain electrostatic repulsion and allows attractive forces of hydrogen bonding, hydrophobic and van der Waals interactions to cause polymer precipitation at a pH near 6.3 to 6.4. Admixing a polyol-phosphate dibasic salt (i.e. glycerol-phosphate) to an aqueous solution of chitosan can increase the pH of the solution while avoiding precipitation. In the presence of these particular salts, chitosan solutions of substantial concentration (0.5-3%) and high molecular weight (>several hundred kDa) remain liquid, at low or room temperature, for a long period of time with a pH in a physiologically acceptable neutral region between 6.8 and 7.2. This aspect facilitates the mixing of chitosan with cells in a manner that maintains their viability. An additional important property is that such chitosan/polyol-phosphate (C/PP) aqueous solutions solidify or gel when heated to an appropriate temperature that allows the mixed chitosan/cell solutions to be injected into body sites where, for example cartilage nodules can be formed in subcutaneous spaces.
Chitosan is thus recognized as a biodegradable, biocompatible, antibacterial and haemostatic biopolymer that is able to promote wound healing, drug absorption, and tissue reconstruction. Due to the above mentioned intrinsic properties, chitosan also has been widely explored in numerous cosmetic and pharmaceutical applications. Therefore, considering the great potential of chitosan, there is a continuous need to improve the properties of known thermosensitive chitosan hydrogels which are still considered as very promising for a wider range of biomedical applications.
U.S. Pat. No. 6,344,488, discloses a pH-depend temperature controlled chitosan composition prepared by neutralizing a commercial chitosan having a deacetylation degree ranging from 70 to 95% with mono-phosphate dibasic salts of polyols or sugars, phosphorylated polyols or phosphorylated sugars, exemplified in particular with β-glycerophosphate (β-GP). Because of its unique properties, the thermogelling chitosan-GP system has raised significant biomedical interest. However, high concentration of β-GP was required, particularly for chitosan having DDA between 70 and 85%, in order to achieve fast gelation at body temperature and to avoid rapid elimination of the hydrogel after its administration (Chenite et al., 2000, Biomaterials, 21: 2155-2161; and Chenite et al., 2001, Carbohydrate Polymers, 46: 39-47). This resulted into very high osmolarity, more than twice of that of physiological extracellular fluid (Crompton et al., 2007, Biomaterials, 28: 441-449; and Hoemann et al., 2005, Osteoarthritis Cartilage, 13: 318-329). Ideally, the hydrogel should be isotonic with the extracellular fluid; and its osmolarity should be around 300 mOsm. The osmolarity is a very important factor regulating biocompatibility of the hydrogel with cells either in vitro or in vivo.
Further, in an attempt to improve the gelation properties of chitosan-GP system, particularly for isotonic compositions, U.S. patent application publication No. 2009/0202430 proposed the addition of glyoxal as chemical crosslinker. In another description, particular composition of chitosan-GP system has been combined with blood in the attempt to improve and stabilize blood clots (U.S. Pat. No. 7,148,209 and U.S. patent application publication No. 2010/0178355).
U.S. patent applications Nos. 2009/0270514 and 2010/0113618 described the preparation of thermogelling chitosan solutions by using, instead of β-GP, either (NH4)2HPO4 solution or NaOH solution respectively. However, the use of ammonium phosphate salts or all the salts derived from organic bases as disclosed in U.S. patent applications No. 2009/0270514 may be harmful or damageable to cells and living tissues, even if they are at a concentration which normally leads to isotonic thermogelling chitosan solutions. U.S. patent applications No. 2010/0113618 was restricted to reacetylated chitosan having a degree of deacetylation (DDA) ranging from 30 to 60%. Moreover, the NaOH solution is beforehand added with high concentration of 1,3-propanediol, an organic reagent which can be potentially toxic to cells and living tissues. Despite the slight improvement provided by the use polyoses or polyols instead 1,3-propanediol, as disclosed in U.S. patent applications No. 2009/0004230, the toxicity problem remain unsolved, so the system can not be a suitable matrix for cells, sensitive proteins or living tissues.
It is also well known that a solution of bicarbonate salt as NaHCO3, a weak base, can be used to increase the pH of chitosan solution in the vicinity of 6.5 without causing any precipitation, but the resulting solution is unable to turn into homogeneous hydrogel in temperature range between 0 and 50° C. In fact, a pseudo-gelation can be observed, occurring at the surface of the solution caused by the release of CO2, as has been reported by recent study (Liu et al., 2011, Int. J. Pharm., 414: 6-15). In such a case, to achieve gelation of the whole sample, it is necessary to disturb the solution and bring ungelled solution to the surface from the bottom of the sample. This leads to non homogeneous hydrogel.
Thus, there is still a need to be provided with an improved thermogelling chitosan solution having better biocompatibility properties, that is not toxic to cells and living tissue.